CN113748210A - Microorganism for producing dicarboxylic acid and method for producing dicarboxylic acid using the same - Google Patents

Abstract

The present invention relates to a candida tropicalis cell line comprising a mutant gene with improved tolerance to matrix cell cytotoxicity and to a method for producing dicarboxylic acids using the candida tropicalis cell line. The dicarboxylic acid-producing candida tropicalis cell line developed according to the present invention has improved tolerance to the existing substrate toxicity, remarkably improved dicarboxylic acid production efficiency, and thus can be used for the biological production of dicarboxylic acid, and is expected to have high industrial applicability, as compared to the existing cell line.

Description

Microorganism for producing dicarboxylic acid and method for producing dicarboxylic acid using the same

Technical Field

The present invention relates to a microorganism having improved tolerance to cytotoxicity of a substrate, including a mutant gene, and a method for producing dicarboxylic acid (DCA) using the same.

Background

Dicarboxylic acids (DCA) are organic compounds containing two carboxyl groups (-COOH). The dicarboxylic acids of the general formula HO₂C-R-CO₂H, wherein R may be an aliphatic or aromatic group. Generally, dicarboxylic acids exhibit chemical reactions and reactivities similar to monocarboxylic acids. Dicarboxylic acids are also used to prepare copolymers, such as polyamides and polyesters. The dicarboxylic acid most widely used in industry is adipic acid, which is a precursor for the production of nylon. Other examples of dicarboxylic acids include aspartic acid and glutamic acid, which are two amino acids in the human body. In addition, other carboxylic acids have been used in various industrial fields.

Such dicarboxylic acids have been prepared by chemical processes or biological methods. As an example of the preparation of dicarboxylic acids, sebacic acid, which is one of dicarboxylic acids, can be synthesized even using phenol and cresol, but castor oil oxidation is known to be the most environmentally friendly and most price competitive method. Castor oil is transesterified by steam cracking and ricinoleic acid is produced by transesterification. Ricinoleic acid thus produced is heated at 250 ℃, mixed with an alkali (e.g., molten caustic soda, etc.), and decomposed into octanol (2-octanol) and sebacic acid by caustic digestion. The product thus produced was purified to obtain high-purity sebacic acid (U.S. patent nos. 5,952,517 and 6,392,074). However, this method has disadvantages in that it requires a high temperature treatment at 300 ℃ or more to achieve the above object, and uses a strong acid such as sulfuric acid, and generates a large amount of environmental pollutants due to the use of materials such as heavy metals, toxic organic solvents, etc. In addition to the chemical method for preparing sebacic acid, this production can be performed by electrolysis of Potassium monoethyl adipate (Potassium monoethyl adipate).

In previous studies, it has been reported that dicarboxylic acid is produced biologically using a strain of Candida tropicalis (Candida tropicalis) which is excellent in ω -oxidation ability and blocked in β -oxidation. However, this method is limited in that it cannot efficiently produce dicarboxylic acids because Candida tropicalis strains have poor tolerance to substrates exhibiting cytotoxicity (non-patent document 1: David L. craft et al, applied and environmental microbiology, 69(10): 5983-. In particular, Korean patent (patent application No. 10-2015-0149253) discloses the production of sebacic acid from a cytotoxic substrate using a mutant strain of Candida tropicalis, but there are no reports on studies on tolerance-enhancing factors and sebacic acid production pathways. Therefore, it is important to develop a useful strain capable of mass-producing dicarboxylic acids using a biological method.

Accordingly, the present inventors screened strains having enhanced tolerance to substrates having cytotoxicity to exhibit enhanced capability of producing dicarboxylic acids by an evolutionary method using a dicarboxylic acid-producing Candida tropicalis strain, and identified genes from the Candida tropicalis strain having an effect on substrate tolerance. Therefore, the present invention has been completed based on these facts.

[ related Art document ]

[ patent documents ]

Korean patent laid-open publication No. 10-2017-0048763

[ non-patent document ]

Craft et al, applied and environmental microbiology, 69(10):5983-5991,2003

Disclosure of Invention

Technical problem

It is therefore an object of the present invention to provide a strain of candida tropicalis with improved tolerance to cytotoxicity of a substrate, wherein the strain comprises a mutation in one or more genes selected from the group consisting of: consisting of SEQ ID NO: 1, the LIP1 (lipase) gene represented by the base sequence shown in SEQ ID NO: 2 and a FAT1 (fatty acid transport) gene represented by the base sequence shown in SEQ ID NO: 3, or wherein the strain is transformed with one or more mutant genes selected from the group consisting of: a mutated LIP1 gene, a mutated FAT1 gene, and a mutated MRP1 gene.

Another aspect of the present invention is to provide a method for producing a dicarboxylic acid by culturing a Candida tropicalis strain with a substrate.

Technical scheme

In order to achieve the above object, the present invention provides a candida tropicalis strain having improved tolerance to cytotoxicity of a substrate, wherein the strain comprises a mutation in one or more genes selected from the group consisting of: consisting of SEQ ID NO: 1, the LIP1 (lipase) gene represented by the base sequence shown in SEQ ID NO: 2 and a FAT1 (fatty acid transport) gene represented by the base sequence shown in SEQ ID NO: 3, or wherein the strain is transformed with one or more mutant genes selected from the group consisting of: a mutated LIP1 gene, a mutated FAT1 gene, and a mutated MRP1 gene.

According to one embodiment, when a normal Candida tropicalis strain is cultured in a medium containing a substrate exhibiting cytotoxicity to screen for strains that have excellent viability on the substrate in terms of evolution, it has been found through genomic analysis of the screened strain that one or more endogenous genes selected from the group consisting of: consisting of SEQ ID NO: 1, the LIP1 (lipase) gene represented by the base sequence shown in SEQ ID NO: 2 and a FAT1 (fatty acid transport) gene represented by the base sequence shown in SEQ ID NO: 3, and MRP1 (multidrug resistance protein) gene represented by the base sequence shown in the figure. Furthermore, it has been found that when the mutant gene is isolated and transduced into a normal Candida tropicalis strain alone, the Candida tropicalis strain has improved tolerance to substrates exhibiting cytotoxicity.

Consisting of SEQ ID NO: the base sequence of mutant gene mtLIP1 (lipase) of LIP1 (lipase) gene represented by the base sequence shown in 1 can be shown as SEQ ID NO: 4, represented by SEQ ID NO: the base sequence of mutant gene mtFAT1 (fatty acid transport) of FAT1 (fatty acid transport) gene represented by the base sequence shown in 2 can be shown as SEQ ID NO: 5, or by the sequence shown in SEQ ID NO: the base sequence of mutant gene mtMRP1 (multidrug resistance protein) of MRP1 (multidrug resistance protein) gene represented by the base sequence shown in 3 can be shown as SEQ ID NO: and 6.

One or more mutant genes may be included in the vector. The vector may be in a form in which the genes may be operably linked. In the present invention, the term "operably linked" generally means that a base expression control sequence is operably linked to a base sequence encoding a desired protein to perform its function, thereby effecting the expression of the base sequence encoding the desired protein. Operable linkage of the vectors can be achieved using genetic recombination techniques known in the art, and site-specific DNA digestion and linkage can be performed using digestive enzymes and ligases and the like known in the art.

In the present invention, the term "vector" refers to any medium used for cloning and/or transferring bases into a host cell. The vector may be a replicon capable of binding to another DNA segment to replicate the bound segment. The term "replicon" refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus) that functions in vivo as an autologous unit of DNA replication, that is, that can replicate through its own regulation. The term "vector" may include viral and non-viral media for introducing bases into a host cell in vitro, ex vivo or in vivo. In addition, the term "vector" may include microspherical DNA. For example, the vector may be a plasmid without a bacterial DNA sequence. The term "vector" may also include transposons, such as Sleeping Beauty (Izsvak et al. J. MoI. biol.302:93-102(2000)), or artificial chromosomes. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, xii, ASHII, APII, t10, t11, Charon4A, Charon21A, and the like can be used as a phage vector or cosmid vector. Plasmid vectors may also be used. The vector that can be used in the present invention is not particularly limited, and a known expression vector can be used.

The Candida tropicalis strain may express the mutant gene, or may include a vector containing the mutant gene.

Candida tropicalis strains are strains in which the beta-oxidation pathway is blocked. In particular, the Candida tropicalis strain may be a strain in which the β -oxidation pathway is blocked, thereby producing a dicarboxylic acid using a substrate.

The substrate may be Fatty acid methyl ester (FATTy acid methyl ester, FAME). In this case, the substrate exhibits cytotoxicity against the dicarboxylic acid-producing Candida tropicalis strain. In particular, the fatty acid methyl ester may be a fatty acid methyl ester comprising C₆-C₂₀One of fatty acid methyl esters of alkylene groups. More particularly, the fatty acid methyl ester may be Decanoic Acid Methyl Ester (DAME).

According to one embodiment of the present invention, the following candida tropicalis strains were demonstrated to have improved tolerance to cytotoxicity of fatty acid methyl esters, thereby exhibiting excellent survival ability in substrates: wherein one or more is selected from the group consisting of SEQ ID NO: 1, the LIP1 (lipase) gene represented by the base sequence shown in SEQ ID NO: 2 and a FAT1 (fatty acid transport) gene represented by the base sequence shown in SEQ ID NO: 3, or a candida tropicalis strain in which one or more mutant genes are introduced.

According to another aspect, the present invention provides a method for producing dicarboxylic acid (DCA), the method comprising culturing a candida tropicalis strain with improved tolerance to cytotoxicity of a substrate, in which one or more selected from the group consisting of SEQ ID NOs: 1, the LIP1 (lipase) gene represented by the base sequence shown in SEQ ID NO: 2 and a FAT1 (fatty acid transport) gene represented by the base sequence shown in SEQ ID NO: 3, or introducing one or more mutant genes into the candida tropicalis strain.

Since the method for producing a dicarboxylic acid according to the present invention actually uses the above-mentioned Candida tropicalis strain, a description of the common contents between the two is omitted so as not to unduly complicate the present specification.

The Candida tropicalis strain may be a strain in which the beta-oxidation pathway is blocked.

The substrate required for dicarboxylic acid production by a Candida tropicalis strain may be Fatty Acid Methyl Ester (FAME). In this case, the substrate exhibits cytotoxicity against the dicarboxylic acid-producing Candida tropicalis strain. In particular, the fatty acid methyl ester may be a fatty acid methyl ester comprising C₆-C₂₀One kind of fatty acid methyl ester with alkyl chain.

According to one embodiment of the present invention, a mutant strain obtained by introducing one or more genes selected from the group consisting of: consisting of SEQ ID NO: 1, mutant gene mtLIP1 (lipase) of LIP1 (lipase) gene represented by the base sequence shown in SEQ ID NO: 2, and a mutant gene FAT1 (fatty acid transport) of FAT1 (fatty acid transport) gene represented by the base sequence shown in SEQ ID NO: 3, and a mutant gene mtMRP1 (multidrug resistance protein) of MRP1 (multidrug resistance protein) gene represented by the base sequence shown in figure 3. The dicarboxylic acid-producing abilities of the mutant Candida tropicalis strains and the Candida tropicalis strains in which the beta oxidation pathway is blocked were compared. The results confirmed that the mutant Candida tropicalis strains of the present invention have excellent dicarboxylic acid-producing ability, which indicates that the resistance of Candida tropicalis strains to the cytotoxicity of substrates is improved by mutation of the disclosed genes, which results in improved strain viability.

Advantageous effects

The candida tropicalis strain for dicarboxylic acid production developed according to the present invention has improved tolerance to existing toxic substrates and significantly improved dicarboxylic acid production efficiency compared to existing strains, and thus is expected to have high industrial applicability because it is suitable for use in a biological process for producing dicarboxylic acid.

Drawings

FIG. 1 shows the results of comparing the cytotoxicity of methyl Decanoate (DAME), Decanoic Acid (DA) and Sebacic acid (Sebacic acid, SA) against Candida tropicalis strains;

FIG. 2 shows a growth diagram of a mutant Candida tropicalis strain according to the following generations: e1 (generation 170), E2 (generation 470), E4 (generation 700), E5 (generation 720), and ES 5;

FIG. 3 shows the measurement results of the Dry Cell Weight (DCW) of each of the mutant Candida tropicalis strains (WT, E5 and ES 5);

FIG. 4 shows the results of analysis of DAME consumption in the medium of each of mutant Candida tropicalis strains (WT, E5 and ES 5);

FIG. 5 shows the results of an analysis of sebacic acid production of each of mutant Candida tropicalis strains (WT, E5, and ES 5);

FIGS. 6 to 8 show the results of comparing and analyzing the dry cell weight (FIG. 6), DAME consumption (FIG. 7), and sebacic acid production (FIG. 8) of a mutant Candida tropicalis strain (mtSAP1) into which mtLIP1 gene was introduced, a strain (β -KO) in which β -oxidation pathway was deleted as a parent strain, and a strain (SAP1) into which LIP1 gene was introduced;

fig. 9 to 11 show the results of comparing and analyzing the dry cell weight (fig. 9), the dam consumption (fig. 10), and the sebacic acid production (fig. 11) of a mutant candida tropicalis strain (mtSAP2) into which mtFAT1 gene was introduced, a strain (β -KO) in which β -oxidation pathway was deleted as a parent strain, and a strain (SAP2) into which FAT1 gene was introduced;

fig. 12 to 14 show the results of comparing and analyzing the dry cell weight (fig. 12), the dam consumption (fig. 13), and the sebacic acid production (fig. 14) of a mutant candida tropicalis strain (mtSAP3) into which mtMRP1 gene was introduced, a strain (β -KO) in which β -oxidation pathway was deleted as a parent strain, and a strain (SAP3) into which MRP1 gene was introduced;

FIG. 15 is a schematic diagram of a plasmid vector for transducing Candida tropicalis, which contains mtLIP1 gene, mtFAT1 gene and mtMRP1 gene;

FIGS. 16 to 18 show the results of analysis of the dry cell weight (FIG. 16), DAME consumption (FIG. 17), and sebacic acid production (FIG. 18) of a mutant Candida tropicalis strain into which two or more genes among mtLIP1 gene, mtFAT1 gene, and mtMRP1 gene were introduced;

FIG. 19 shows confirmation of the mutant Candida tropicalis strain (mtSAP7) and parent strain (. beta. -KO) for C by measuring the dry cell weight and dicarboxylic acid production amount of the mutant Candida tropicalis strain (mtSAP7) and parent strain (. beta. -KO) into which mtLIP1 gene, mtFAT1 gene and mtMRP1 gene were introduced₁₀Results of cytotoxic tolerance of Fatty Acid Methyl Ester (FAME) substrates;

FIG. 20 shows confirmation of the mutant Candida tropicalis strain (mtSAP7) and parent strain (β -KO) for C by measuring the dry cell weight and dicarboxylic acid production amount of the mutant Candida tropicalis strain (mtSAP7) and parent strain (β -KO) introduced with mtLIP1 gene, mtFAT1 gene and mtMRP1 gene₈To C₁₂Results of cytotoxic tolerance of Fatty Acid Methyl Ester (FAME) substrates.

Detailed Description

The configuration and effects of the present invention will be described in more detail below with reference to embodiments of the present invention. However, it should be understood that the embodiments described herein are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

EXAMPLE 1 confirmation of biological cytotoxicity of substrate and product

To confirm the cytotoxicity of methyl Decanoate (DAME) and its product (i.e., sebacic acid) used as a substrate for producing sebacic acid, toxicity tests were performed under the following conditions. More specifically, the Candida tropicalis MYA-3404 strain used in the related art for the production of sebacic acid was cultured in YNB medium (10g/L yeast extract and 20g/L peptone) to which DAME, DA or sebacic acid was added at a concentration of 5 g/L. The culture temperature was 30 ℃ and the strain was cultured at 200rpm for 36 hours.

As a result, as shown in FIG. 1, it was confirmed that the strain grew at a slower growth rate or did not grow in the medium to which DAME, DA or sebacic acid was added, compared to the control to which DAME, DA or sebacic acid was not added. More specifically, it was confirmed that the growth rate and total cell amount of the strain were reduced in the medium to which sebacic acid was added, compared to the control, and that the strain did not grow in the medium to which DAME or DA was added at a concentration of 5 g/L. From the results, it was confirmed that all the substrates (DAME and DA) and the product (sebacic acid) were cytotoxic. Among them, DAME was confirmed to have stronger cytotoxicity compared with sebacic acid. From the above results, it was confirmed that in order to produce sebacic acid at a high concentration, it is preferable to develop a strain having tolerance to DAME as a substrate.

EXAMPLE 2 development of DAME-resistant Strain Using evolutionary engineering method

In order to develop a strain tolerant to DAME, which is a substrate having cytotoxicity, Candida tropicalis (C.tropicalis) MYA-3404 strain was cultured in YNB medium (10g/L yeast extract and 20g/L peptone) supplemented with DAME at a concentration of 10 g/L. In this case, it was confirmed that the concentration of DAME in the medium was maintained at about 0.45g/L (maximum solubility) due to the low solubility of the DAME substrate (confirmed by the results of the internal experiment). The growth curve of the inoculated strain was determined by measuring the absorbance at a wavelength of 600 nm.

And (3) observing the absorbance of the culture medium inoculated with the strain in real time, and then subculturing the strain in a fresh culture medium until the growth of the strain reaches a metaphase exponential phase. Specific growth rates were calculated from the measured absorbance values, and strains at stages where the specific growth rates varied greatly were determined as E1 (at 170 generations), E2 (at 470 generations), E4 (at 700 generations) and E5 (at 720 generations), respectively. Further, the E5 strain obtained by the above method was subcultured in YNB medium (10g/L yeast extract and 20g/L peptone) supplemented with 20g/L glucose as a non-toxic carbon source, and then re-inoculated in a DAME substrate to screen for a strain that maintains tolerance to DAME even after replacement of the carbon source, and this strain was named "ES 5".

The growth curve of the mutant strain was determined. The results confirmed that the specific growth rate of the mutant strain increased as the subculture proceeded, as shown in FIG. 2. It was confirmed that the ES5 strain also exhibited a high specific growth rate without losing its tolerance and had constant tolerance to the DAME substrate. To more specifically determine the specific growth rate and the tolerance to the DAME substrate, the WT strain as a control and the E5 and ES5 strains as mutant strains were cultured in YNB medium supplemented with DAME at a concentration of 10 g/L. The incubation temperature was 30 ℃ and the incubation time was 120 hours, and samples were collected every 12 hours or every 24 hours to measure the Dry Cell Weight (DCW) of the samples.

The Dry Cell Weight (DCW) of each strain was measured. As a result, as shown in FIG. 3, it was confirmed that the WT strain had a very low DCW value (no growth), whereas the E5 and ES5 strains had the maximum cell mass after 120 hours of strain culture, and the cell masses of the ES5 and E5 strains were increased to 2.5g/L and 2.2g/L, respectively. Based on the results, it was confirmed that the mutant strains E5 and ES5 obtained by the evolutionary engineering method were tolerant to DAME substrates, resulting in a greater degree of growth.

Example 3 confirmation of phenotypic changes of the parent Strain (WT) and the mutant Strain (E5 and ES5)

The actual amounts of the DAME substrate consumed and the amount of sebacic acid produced by the mutant strains E5 and ES5 obtained in example 2 were compared with the parent strain (WT). To determine the DAME substrate consumption and sebacic acid productivity, each of the WT, E5 and ES5 strains was cultured at 30 ℃ for 120 hours in YNB medium supplemented with DAME at a concentration of 10 g/L.

Samples for analysis were collected every 12 hours or every 24 hours and the concentrations of DAME and sebacic acid in the medium were analyzed using Gas chromatography/Mass spectrometry (GC/MS). The GC/MS conditions are listed in Table 1 below.

[ Table 1]

GC/MS analysis samples for DAME analysis were prepared as follows. 4mL of the collected culture broth was mixed with 1mL of 10M HCl and vortexed for one minute. An equal amount of hexane was added to the mixture and incubated at room temperature for 10 minutes. After 10 minutes, the mixture was mixed well by vortexing and then centrifuged at 12000rpm for 1 minute. The supernatant (hexane) was collected from the mixed solution separated into two layers for GC/MS analysis. As with the previous DAME analysis of the collected samples, 10M HCL was added to the sample for analysis of sebacic acid, and then mixed. Thereafter, an equal amount of ethyl acetate was added thereto and mixed. Then, the ethyl acetate layer was collected and completely dried using a vacuum evaporator. Subsequently, 50. mu.L of pyridine (Sigma-Aldrich, StLouis, MO, USA) was added to 2% (w/v) concentration of O-methylhydroxylamine hydrochloride (Sigma-Aldrich, StLouis, MO, USA), followed by hydroxamation at 75 ℃ for 30 minutes. Then, 80. mu.L of N-methyl-N- (trimethylsilyl) trifluoroacetamide (Sigma-Aldrich, StLouis, MO, USA) was added thereto, followed by derivatization at 40 ℃ for 30 minutes. For quantification of the results, DAME and sebacic acid (Sigma-Aldrich, StLouis, MO, USA) were purchased and diluted in certain proportions. Then, a sample for analysis was prepared in the same manner as described above, and then analyzed by GC/MS. The collected samples were analyzed by GC/MS to measure the amount of DAME in the medium. As a result, as shown in FIG. 4, it was confirmed that the amount of DAME was not greatly reduced in the medium inoculated with the parent strain, whereas the amount of substrate was drastically reduced in the medium inoculated with the E5 and ES5 strains. Thus, it was confirmed that DAME was present at about 3.1g/L in the medium inoculated with E5 strain and about 2.8g/L in the medium inoculated with ES5 strain 120 hours after the strain reached the maximum cell mass. Furthermore, the sebacic acid production of the E5 and ES5 strains (fig. 5) was very different from that of the parent strain. In this case, it was confirmed that the yield of sebacic acid of the parent strain after 48 hours of fermentation was about 44.3mg/L, and the yields of sebacic acid of the E5 strain and the ES5 strain were about 177.4g/L and about 218.4mg/L, respectively. Therefore, the fact that the E5 and ES5 strains exhibited high DAME substrate consumption and sebacic acid productivity was judged to be due to mutations in the strains when they were subcultured with DAME, a substrate having biotoxicity. Therefore, base sequencing and transcriptome analysis were performed on the ES5 strain that exhibited the highest DAME consumption and sebacic acid productivity.

EXAMPLE 4 transcriptome analysis of DAME-tolerant mutant (ES5)

To examine the changes in transcriptome in the medium with and without DAME, the transcriptome of ES5 strain grown in the medium supplemented with DAME and ES5 strain grown in the medium without DAME was analyzed.

The ES5 strain was cultured at 30 ℃ for 24 hours in YNB medium containing no DAME and in YNB medium supplemented with 10g/L DAME. The cultured cells were collected and washed with water. Thereafter, the collected cells were used as a sample for whole RNA extraction. RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and the concentration and purity of the extracted RNA was measured using NanoDrop (Thermo Scientific, Wilmington, DE, USA) and Agilent Bioanalyzer 2100(Santa Clara, Ca, USA), respectively.

The transcriptome of the mutant ES5 strain was analyzed and compared to that of the parental strain. The results demonstrated a total of 453 genes up-regulated in the ES5 strain compared to the parental strain and 147 genes down-regulated in the ES5 strain compared to the parental strain. Details of the number of genes and clustering are detailed in table 2.

[ Table 2]

Results of comparison/analysis of transcriptome of parent Strain and DAME tolerant mutant (ES5)

EXAMPLE 5 sequencing of all bases of DAME-tolerant mutant Strain (ES5) and search for candidate genes associated with improvement of tolerance

In order to identify genes associated with the improvement in DAME tolerance of ES5 strain obtained by the evolutionary engineering method, ES5 strain was subjected to full-base sequencing. Extraction of genomic DNA for full base sequencing was performed using a DNA isolation kit (Epicentre, Madison, Wis., USA). The entire base sequence was analyzed using the Illumina Hiseq 2500NGS platform (DNA Link USA, inc., San Diego, CA, USA).

A total of 13,256,614 reads were obtained by full base sequencing, covering approximately 87.98% of the full base sequence, and then aligned using the Picard tool 1.128 software. The aligned sequences were annotated with SNPEff 4.1(GRCh 37.75) and the spectra were plotted using BWA7.12 software. In this case, the SNP DB is deleted by the dbSNP138 software. Finally, the mutated genes were identified by comparing the genes obtained by the NCBI, Uniprot, KEGG databases.

As a result, it was confirmed that mutations occurred in a total of 770 genes, and a total of 106 mutant genes excluding genes whose functions were not identified were obtained. Among them, genes LIP1 (lipase, Unit.ID: C5M8S7), FAT1 (fatty acid transporter, Unit.ID: C5M964), MRP1 (multidrug resistance protein CDR1, Unit.ID: C5M804) expected to be involved in improvement of tolerance to cytotoxic substrates and associated with increased production of sebacic acid were selected and named LIP1(SEQ ID NO: 1), FAT1(SEQ ID NO: 2) and MRP1(SEQ ID NO: 3), respectively. Their mutant genes were named mtLIP1(SEQ ID NO: 4), mtFAT1(SEQ ID NO: 5) and mtMRP1(SEQ ID NO: 6). The mutation sites of each gene are listed in tables 3, 4 and 5.

[ Table 3]

LIP1 gene (Seq _1-LIP1, Seq _2-mtLIP1)

[ Table 4]

FAT1 gene (Seq _1-FAT1.Seq _2-mtFAT1)

[ Table 5]

MRP1 gene (Seq _1-MRP1.Seq _2-mtMRP1)

[ example 6] preparation of Strain overexpressing mtLIP1, mtFAT1 and mtMRP1 genes and phenotypic Change

[ Table 6]

Primer list for cloning tolerance genes

Restriction sites are underlined.

[ Table 7]

Plasmids used in this study

[6-1] preparation of Strain overexpressing LIP1 Gene and mtLIP1 Gene and phenotypic Change

In order to prepare candida tropicalis strains overexpressing the same (no mutation) gene (LIP1) present in the parent strain and the mutant gene (mtLIP1) selected in example 5, respectively, cloning experiments were performed as follows. For efficient expression of the introduced gene, the ADH promoter (introduced at ADHpro, XhoI/SalI restriction sites) and ADH terminator (introduced at ADHter, XbaI/NotI restriction sites) were amplified using ADHpro _ F/R primer and ADHter _ F/R primer (Table 6), and ADH promoter and ADH terminator were preferentially introduced into pRS420 vector to construct plasmid 6. To obtain the LIP1 gene and mtLIP1 gene, genomic DNA extracted from each of Candida tropicalis MYA _3404 strain and Candida tropicalis ES5 strain was amplified using LIP1-F primer and LIP1_ R primer (Table 5), and the resulting DNA fragments were ligated to the SalI and XbaI restriction sites of plasmid 6 thus constructed. Thus, plasmid 7 into which LIP1 was introduced and plasmid 8 into which mtLIP1 was introduced were finally obtained (Table 7). Then, plasmids 7 and 8 were transformed into Candida tropicalis 20962 deficient in the β -oxidation pathway, and Candida tropicalis _ LIP1 strain and Candida tropicalis _ mtLIP1 strain were finally prepared.

Phenotypic changes of the strains introduced with the LIP1 gene and mtLIP1 gene, respectively, were compared with those of a control (Candida tropicalis strain deficient in β -oxidation pathway (. beta. -KO)). As a result, as shown in FIG. 6, it was confirmed that the growth of the strain introduced with the LIP1 and mtLIP1 genes was improved as compared with the β -KO strain. In particular, it was confirmed that the DCW value (about 1.2g/L) of mtLIP1 gene-introduced strain was the largest. The DAME consumption of the three strains was compared. As a result, it was confirmed that the β -KO strain hardly consumed DAME, while the LIP1 gene-introduced strain and the mtLIP1 gene-introduced strain had higher substrate consumption amounts as compared with the control. In particular, it was confirmed that the mtLIP1 gene-introduced strain consumed about 70% of the total substrate within 120 hours (FIG. 7). Finally, the SA yields of the three strains were compared. As a result, it was confirmed that the β -KO strain and the mtLIP1 gene-introduced strain produced about 300mg/L of sebacic acid, while the mtLIP1 gene-introduced strain produced about 900mg/L of sebacic acid (FIG. 8).

From the above results, it was confirmed that the LIP1 gene in Candida tropicalis strains is a gene having an influence on the growth of Candida tropicalis strains, the consumption of DAME substrate, and the production of sebacic acid, and that the mutant gene mtLIP1 obtained according to the present invention greatly contributes to the increase in sebacic acid production.

[6-2] preparation of mtFAT1 Gene-introduced Strain and confirmation of phenotypic Change

Similarly to example 6-1, phenotypic changes of the strain introduced with the FAT1 gene and the strain introduced with the mtFAT1 gene were compared with that of a control (. beta. -oxidative pathway-deficient Candida tropicalis strain (. beta. -KO)). The DNA fragments amplified from the genomic DNAs of candida tropicalis MYA _3404 strain and candida tropicalis ES5 strain used in example 6-1 using the FAT1_ F primer and the FAT1_ R primer (table 6) were ligated to the SalI and XbaI restriction sites of the pRS420 vector present in plasmid 6 to finally prepare plasmid 9 into which the FAT1 gene was introduced and plasmid 10 into which the mtFAT1 gene was introduced (table 7). The plasmids 9 and 10 thus prepared were then transformed into Candida tropicalis 20962 deficient in the β -oxidation pathway. Finally, the candida tropicalis _ FAT1 strain and candida tropicalis _ mtFAT1 strain were prepared.

As a result, as shown in fig. 9, it was confirmed that there was no significant difference in DCW value between the β -KO strain and the strain introduced with FAT1 gene, but the strain introduced with mtFAT1 gene had a high DCW value (about 1.5g/L), indicating that the growth of cells was significantly improved due to mutation of FAT1 gene. The DAME consumption of the three strains was compared. As a result, it was confirmed that the β -KO strain hardly consumed dam, and the substrate consumption of the strain introduced with the FAT1 gene was not significantly different compared to the β -KO strain, but the strain introduced with the mtFAT1 gene had a relatively high substrate consumption compared to the control. Furthermore, it was confirmed that the strain introduced with mtFAT1 gene consumed about 70% of the total substrate within 120 hours (fig. 10). Finally, the strains were compared for sebacic acid production. As a result, it was confirmed that, after 120 hours of fermentation, the β -KO strain and the mtFAT1 gene-introduced strain produced about 280mg/L and 384mg/L of sebacic acid, respectively, while the mtFATP1 gene-introduced strain produced about 1275mg/L of sebacic acid (FIG. 11).

From the above results, it was confirmed that the FAT1 gene in candida tropicalis strain is a gene related to growth of candida tropicalis strain, consumption of DAME substrate, and production of sebacic acid, similarly to the LIP1 gene, and it was also confirmed that mtFAT1 gene obtained according to the present invention greatly contributes to increase of sebacic acid production.

[6-3] preparation of mtMRP1 Gene-introduced Strain and confirmation of phenotypic Change

Finally, the phenotypic changes of the MRP1 gene-introduced strain and the mtMRP1 gene-introduced strain were compared with those of a control (Candida tropicalis strain deficient in the. beta. -oxidation pathway (. beta. -KO)). As in the previous examples, the vector used for cloning was the pRS420 vector into which ADHpro and ADHter for the construction of plasmid 6 were introduced. The vector was amplified using MRP1_ F primer and MRP1_ R primer, and then ligated to SalI/XhoI restriction sites. In this way, plasmid 11 and plasmid 12 were constructed, and the constructed plasmids were transformed into candida tropicalis 20962 deficient in β -oxidation pathway to finally prepare candida tropicalis _ MRP1 strain and candida tropicalis _ mtMRP1 strain.

The MRP1 gene-introduced strain and mtMRP1 gene-introduced strain prepared by the above method were compared with the β -KO strain used as a control. As a result, it was confirmed that the growth of the MRP1 gene-introduced strain and the mtMRP1 gene-introduced strain was improved. In particular, it was confirmed that the mtMRP1 gene-introduced strain had a high cell count, about 1.5g/L (FIG. 12). The DAME consumption of the three strains was compared. As a result, it was confirmed that the β -KO strain consumed about 10% of the substrate after 120 hours, whereas the MRP1 gene-introduced strain consumed about 40% of the substrate, and that the mtMRP1 gene-introduced strain consumed more than about 9g/L of DAME, based on the initial amount of DAME of 10g/L (FIG. 13). The sebacic acid yields of the three strains were compared. As a result, it was confirmed that the β -KO strain and the MRP1 gene-introduced strain produced about 280mg/L and about 488mg/L of sebacic acid, respectively, while the mtMRP1 gene-introduced strain produced about 1677mg/L of sebacic acid, indicating that the production of sebacic acid was increased about 6-fold in the mutant strain compared to the parent strain (FIG. 14).

From the above results, it was confirmed that the introduced MRP1 gene and mtMRP1 gene induced phenotypic changes of the strain, and that these genes had a positive effect on improving sebacic acid productivity.

[6-4] preparation of a Strain (Candida tropicalis mtSAP7) producing sebacic acid in Large quantities and production of sebacic acid by high-Density culture

A strain (Candida tropicalis mtSAP7) producing sebacic acid in large quantities was prepared, which introduced all mtLIP1, mtFAT1 and mtMRP1 genes whose effects were confirmed in the previous examples. In order to efficiently express three introduced genes, three pairs of ADH promoters (ADHpro1, ADHpro2, and ADHpro3) for promoting the expression of each gene were introduced together with ADH terminators (ADHter1, ADHter2, and ADHter 3). More specific methods for producing the strain are as follows.

(1) The DNA fragment (ADHpro1) amplified from the pAUR123 vector by PCR using the P1_ F primer and the P1_ R primer was ligated to the BamHI/SAlI restriction enzyme site of pET21a vector selected for cloning using T4DNA ligase to construct plasmid 13. For PCR, Q5High-Fidelity Master mix (BioLabs, Ipshich, MA, USA) was used and the same reagents were used in all subsequent experiments.

(2) Meanwhile, a DNA fragment (ADHpro2) amplified from the pAUR123 vector by PCR using the P2_ F primer and the P2_ R primer was ligated to the SalI/NotI restriction enzyme site of the pET21a vector. In this case, for the convenience of subsequent introduction into the strain, a PmlI restriction enzyme sequence (CACGTG) was added in order after the SalI sequence of the restriction enzyme of the forward primer to prepare plasmid 14.

(3) Next, a DNA fragment (ADHter1) amplified from the pAUR123 vector using the P3_ F primer and the P3_ R primer was ligated to the SalI/Btrl restriction enzyme site of plasmid 14 to prepare plasmid 15.

(4) To prepare plasmid 16, previously prepared plasmid 15 was used as a backbone. A DNA fragment (ADHpro1) amplified using plasmid 13 as a template and a P1_ F primer and a P1_ R primer was ligated to the BamHI/SalI restriction site of plasmid 15 to prepare plasmid 16.

(5) The DNA fragment (mtLIP1) amplified from the genomic DNA of Candida tropicalis ES5 strain using the P4_ F primer and the P4_ R primer was ligated to the SalI restriction site between ADHpro1 and ADHter1 of plasmid 16, thereby preparing plasmid 17.

(6) To introduce mtFAT1 gene, mtFAT1 fragment was obtained by PCR using genomic DNA of Candida tropicalis ES5 strain as template and P5_ F primer and P5_ R primer. The mtFAT1 fragment was ligated to the NotI restriction site of plasmid 17 to prepare plasmid 18. Furthermore, the amplified mtFAT1 fragment was subsequently introduced into the NotI restriction site of plasmid 15 to prepare a final plasmid, which was designated "plasmid 19".

(7) To introduce additional promoter and terminator, the ADHter2 fragment and ADHpro3 fragment were obtained by PCR using plasmid 15 as a template and P6_ F primer and P6_ R primer. The ADHter2 fragment and ADHpro3 fragment were then ligated to the NotI/XhoI restriction site of the novel pET21a vector to obtain plasmid 20. In this case, a PmlI restriction site was added before the XhoI restriction site of the P6_ R primer, and then the resulting construct was used to subsequently prepare plasmid 21.

(8) Plasmid 21 was constructed by ligating the DNA fragment (ADHter3) amplified from the pAUR123 vector using the P7_ F primer and the P7_ R primer to the PmlI/XhoI restriction enzyme site of plasmid 20 previously prepared.

(9) Finally, in order to introduce mtMRP1 gene as the third gene, plasmid 22 was constructed by ligating a PCR fragment (mtMRP1) amplified using genomic DNA of candida tropicalis ES5 strain as a template and using P8_ F primer and P8_ R primer to the PmlI restriction site of plasmid 21.

(10) Finally, the restriction fragments of plasmids 18 and 22 previously prepared were fused to prepare plasmid 24, into which plasmid 24 all mtLIP1, mtFAT1 and mtMRP1 genes were introduced. Then, plasmid 25 was constructed by performing PCR using plasmid 24 as a template and P9_ F primer and P9_ R primer and ligating the DNA fragment to the BamHI/XhoI restriction site of pRS 420. The constructed plasmid 25 was transformed into candida tropicalis 20962 deficient in β -oxidation pathway to finally prepare candida tropicalis _ mtSAP7 strain. The construction of the final plasmid 25 and the restriction enzymes used are shown in FIG. 15.

Candida tropicalis strains (mtSAP4(mtLIP1+ mtMRP1), mtSAP5(mtLIP1+ mtFAT1), and mtSAP6(mtMRP1+ mtFAT1)) into which two genes among mtLIP1, mtFAT1, and mtMRP1 were introduced were also prepared based on the above-described method.

The OD changes, substrate consumption and sebacic acid production of the four mutant Candida tropicalis strains were compared. The results confirmed that the candida tropicalis mtSAP7 strain into which all three mutant genes were introduced had excellent cell-forming, substrate-consuming, and sebacic acid-producing abilities (fig. 16, 17, and 18).

In order to examine the effects of the three introduced genes, the prepared Candida tropicalis _ mtSAP7 strain and the Candida tropicalis 20962(β -KO) strain deficient in β -oxidation pathway were fermented under the same conditions. The strain was first cultured in YP medium supplemented with 100g/L of glycerol until the OD of the strain reached 100. After 80 hours of incubation, 200g/L of DAME was added as a substrate, followed by incubation at 30 ℃ for 250 hours.

As a result, no change in OD value was observed in both the candida tropicalis mtSAP7 strain and the candida tropicalis 20962 strain (β -oxidation pathway-deleted strain) used as a control (fig. 19). The SA yields of the two strains were compared. The results confirmed that the strain used as a control had the greatest production of sebacic acid (about 27g/L) at about 250 hours, whereas mtSAP7 strain produced sebacic acid at about 110g/L after about 250 hours had elapsed (FIG. 19).

Based on the above studies, it was confirmed that three genes obtained by all-base sequencing contributed greatly to the improvement of cell formation, substrate-consuming ability and sebacic acid productivity. In addition, it was confirmed that a process having excellent sebacic acid productivity was developed by a high cell density biotransformation process using candida tropicalis mtSAP7 strain, compared to the processes known in the art.

EXAMPLE 7 identification of tolerance of Candida tropicalis mtSAP7 Strain to FAME substrate and dicarboxylic acid production

In addition, using the Candida tropicalis mtSAP7 strain prepared in example 6-4, the ability of the strain to produce sebacic acid from DAME and dicarboxylic acid from various FAME substrates was examined, and then the ability of the Candida tropicalis mtSAP7 strain was compared with the control strain in the same manner as in example 6.

As a result, as shown in FIG. 20, it was confirmed that C is the most important₈To C₁₂C of Candida tropicalis mtSAP7 strain in FAME substrate₈To C₁₂The dicarboxylic acid production is significantly increased. Based on these results, it was confirmed that the mutant candida tropicalis mtSAP7 strain of the present invention exhibited strong tolerance to a FAME substrate having cytotoxicity, and thus greatly contributed to improvement of dicarboxylic acid productivity when FAME was used as a substrate (fig. 20).

Sequence listing

<110> university school labor cooperation group of Korean university

<120> microorganism for producing dicarboxylic acid and method for producing dicarboxylic acid using the same

<130> G21U13C0485P/CN

<150> KR 10-2018-0154372

<151> 2018-12-04

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 1368

<212> DNA

<213> Candida tropicalis

<400> 1

atgagatttc ttgtattcat tacaattatt acatggttga aaactgtatc aactgctcat 60

attcctgcac cacttgctga tccaagtaga gatgagtttt atactccatc tccaggtttt 120

gaatacgcta ctccaggaac tattttaaaa atccgtccaa ctcctcgtgc tgttcgtaat 180

ttattattct ttcatgttcc tttaaaaaac tcttggcaat tgttggttag atctcaagat 240

tcttttggtg aacctaatgc tatagttact acaattcttg aacctatgaa ttcaaatcct 300

tcaaaaattt tatcttatca aacttttgaa gattcaactt cattaaaatg cgctaccagt 360

tataattatc aagttggtat tccaccattt ggaaatgttg ctacccaatt tgaaatgaaa 420

tttataattc ctgctttaaa taaaggatat tttgtaatta gtcctgatta tgaaggacca 480

agaggtgcat ttactgttgg tgcacaagca gcacatgcag tattggattc tattcgtgct 540

gtattgaatt ctgggtctat aacttctatt gatccagatg ctaaagttgc aatgtggggt 600

tattctggag gatccttagc atcaagttgg gcagctgtaa tgcaacctga atatgcacct 660

gaattatcaa ataatttaat aggtgctgcc ttgggaggat ttgttactaa tataactgct 720

gttgctgaat attctgatag aactccactt tctggtcttg ttccagtagc acttaatgga 780

ttagccaatg aatatccatt ggttagacaa ttgcttaatc aagaaataag tcctaaaaaa 840

aatgcaagtt ttcatcgtgg agttcaaaaa tgttttcttc ctgctatagc ttattttaga 900

ggaagaacta ttcttggtag aaataatgaa aagaaagcaa tgtttcctaa tggatggcat 960

ttacttgata atcctgattt ttttgaaatt cttgataaaa ataatttgat ttcttataac 1020

gcaattccaa aaattccaat atttgtatat catggaacaa aagatggagt tgttccgatt 1080

tcctatgctc ataaaatttt cgataaatgg tgtgatgagg gaattgaatc gtttgaattt 1140

gcagaatctt taactactgg acatatattg gaaactttta ctggtgctgc agccgcttgg 1200

acttggttac aaaaacgatt tgatgatgta cctccatata atggttgttt ccatacaaga 1260

agactcacta atttgaagta cacgggagca tcaaagagta taattgatta ttacgatggg 1320

ttgtttaaag aaagcttcac tgtgaagaat agtacctatc ttgtctag 1368

<210> 2

<211> 1953

<212> DNA

<213> Candida tropicalis

<400> 2

atgtcaggat tagaaattgc tgcagctgcc gttcttggta gtcagttatt agaagccaaa 60

tatttaattt ccgatgatgt actgttggcc aaaacagttg ctcttaatgc acttccatat 120

ttatggaaag cctccagggg taaagcttca tattggtatt tctttgaaaa atcagtattt 180

aaaaatccaa ataataaagc attggcattt ccaagaccaa gaaagaatgc accaccacca 240

aaggttgatg atgaaggatt tcaaatttat gacgatcaat ttgacctaga agaatatacc 300

tataaggaat tgtatgacat ggttttgaaa tactcttaca ttttgaaaca tgaatatggt 360

gttactgcaa atgatactat tggtgtttct tgtatgaata aaccactttt cattgtttta 420

tggttggcct tatggaatat tggtgccttg ccagcatttt tgaatttcaa caccaaagat 480

aaaccattga ttcactgtct taaaattgtc aatgctagtc aagttttcgt tgatcctgat 540

tgtgatgctc caatcaaaga tactgaatct caaattaaag aggaattacc acatgttaga 600

ataaattaca ttgatgaatt tgctttgttt gatagattaa gactcaagtc tactccaaaa 660

tacagagctg aagatagtac tagaagacca acagataccg attcttccgc ctgtgcgttg 720

atctatacat caggtaccac tggtttacca aaagcaggta tcatgtcttg gagaaaagca 780

ttcatggctt ctgttttctt tggccatatt atgaaaatta agaatgattc caatgtttta 840

acagctatgc cattgtatca ttcaacagct gctatgttgg gtttgtgtcc tactttaatt 900

gttggtggtt gtgtttctgt ttctcaaaaa ttctcagcca cttcattctg gactcaagct 960

agattatgtg gtgccacaca tattcaatat gttggtgaag tttgtcgtta tttgttaaac 1020

tcaaaacatc acccagatca agatagacac aatgttaaaa ttgcctatgg taatggatta 1080

cgtccagata tatggtctga attcaagaga agattccaca ttgaaggtat tggggaattt 1140

tatgcagcta ctgaatctcc aattgccact acaaacttac aatacggtga atatggtgta 1200

ggtgcctgtc gtaaatatgg ttcacttatt agtttattgt tatctaccca acaaaaattg 1260

gccaagatgg atccagaaga tgaaagtgaa atttataagg atccaaaaac tggattttgt 1320

gttgaagctg catataatga acctggtgaa ttgttgatga gaattttaaa tcctaatgat 1380

attcaaaaat cattccaagg ttattatggt aacaaatctg ctaccaatag caaaattctc 1440

acgaatgttt tcaaaaaagg agatgcttgg tatagaagtg gtgacttgtt gaaaatggat 1500

gaacatcaat tgttgtattt tgttgataga ttgggtgata ccttccgttg gaaatcagaa 1560

aatgtttcag caactgaagt tgaaaatgag ttgatgggat ctaaagcatt gaaacaatct 1620

gttgttgttg gtgttaaagt tccaaatcac gaaggtagag cttgttttgc tgtatgtgaa 1680

gcaaaagatg atttaactca tgaagatatt ttgaaattga ttcatggaca tgttactaaa 1740

tcgttaccag tttatgcaca acctgcattc attaaaatcg gatccattga agcttctcat 1800

aatcataaag ttccaaagaa tcaatttaag aatcaaaaat taccaaaagg tgaagatggt 1860

aaagacttga tttactggtt gaatggtgat aaatatcaag agttgactga agaggattgg 1920

tctttgatct gtactggtaa agccaaattg taa 1953

<210> 3

<211> 4455

<212> DNA

<213> Candida tropicalis

<400> 3

atgggagaaa taaccccaac tgacaaaagc gaagaccagt caatggttaa tgcatatcat 60

ggatttgata ctcatgcatc agaagatata caagatttag ccaaaacttt tactcatcat 120

tcaattggcg atggtactga tggtttacaa agatatctta caaatatgac agaagtacca 180

ggtataaatc cttacaccga agatatttac actagtgacc aattgaatcc agactcagat 240

aattttaatg caaagttttg gatcaagaac ttgagaaaat tgtatgattc agatccagat 300

tattacaagc catcaagatt gggagttgcc tatagagatt taagagctta tggtgtggcc 360

aatgattctg attaccagcc cactgtggca aacgcggtct ggaagtttat caaagaggga 420

ttgcattatt tagaaaaagg tgatggctca aggtattttg atattttaaa atcaatggat 480

ggaataatga aaccaggtga acttacagtt gttttaggta gaccaggggc tggttgttcc 540

acattgttga aaacattggc ttcacaaaca tatggatttc atattggaaa agaatcaaaa 600

atcagttatg atggtttaac tcctcccgaa atcgaaaaaa cttatagggg taatgttgta 660

tactctgcag aaacagatgt tcattttcca catttgactg tcggacaagt cttggaattt 720

gctgctagaa tgagaacgcc acagaacaga ggtgaaggtg tagatagaga aacatatgcc 780

aaacaccttg ctagtgttta tatggctact tatgggttat ctcatacaag aaataccaat 840

gtgggtaacg attttgtcag aggagtttct ggtggtgaaa gaaaaagggt ctccattgct 900

gaagtttcgt tgagtggtgc aaatgttcaa tgttgggata atgccactag aggtttggat 960

gctgcaaccg cattggaatt catcagagca ttgaagactt ctgctgctat tttggaaagt 1020

accccattga ttgctattta tcaatgttca caagatgctt atgacttgtt tgataatgtt 1080

gtcgttttgt atgaaggttt ccaaattttt tttggtaaag ccaataaagc caaggagtat 1140

tttgtaaaca tgggatacaa gtgtcctcaa agacaaacca ctgctgactt tttaacttca 1200

ttgactaatc cagctgaaag agagccatta ccaggttatg agaataaagt cccaaggact 1260

cctcaagaat ttgaagcata ttggaagaaa tccccagagt atactgcatt ggttaatgaa 1320

attgattcat atttcattga gtgtgagaaa ttaaacacca gacaactcta ccaagattca 1380

catgttgcaa gacaatccaa caatattcgt ccatcttcac catatactgt atcatttttc 1440

atgcaagtaa agtatgttat acaaagaaat ttcctccgta tgaaagctga tccatcgatt 1500

ccgttgacta ctattttctc acaactagtt atgggactta ttcttgcctc ggtattttac 1560

aatcttcctg caacttcagg ttctttttac taccgatccg gtgcgcttta ctttggtttg 1620

ttatttaatg ctatttcgtc cctacttgaa attattgccc tttttgaagc aagacccatt 1680

gttgagaaac ataaaaaata tgccctttat cgtccatcag cagatgcatt agcaagtatt 1740

ataagtgagt taccagttaa gttttttcaa tccttgtgtt tcaacattcc tttctatttt 1800

atggttaacc ttagaagaga tgctggtaga ttcttctttt attggttaat tggtatatta 1860

ggtacattca ttatgtcaca cttattcaga tctattggtg cagtatttac tactttagca 1920

ggtgctatga ctccggcggg ggtgatttta ttagcaatga tattatttgc tggatttgtc 1980

attccatttc caagcatgtt gggttggtct aaatggataa aatggataaa tcctgtcact 2040

tatttgtttg aatcacttat ggtaaacgag tatcataata gagagtttga atgcagtgat 2100

ttcgtaccta tgggaccagg atatgagaat cttagtcttg aaaataaggt ttgttcaagt 2160

ttgggtggca tccctggtag tgcttttgtt caaggtgatg attatttaag acttggattt 2220

gccttttcta actcccataa gtggagaaat tttggtatat ctgttgcgtt tgctgtgttt 2280

cttttgtttc tttatgttgc attgactgaa ctcaataaag gtgctatgca aaaaggtgaa 2340

attgtgttgt ttcttagagg atctttgaag aaatacaaga gaaactccag tagcgcagat 2400

attgaatccg gtaaagaaat agtgaaattt aatttccaag acgaagcaga atcttctaat 2460

agtgatcgta ttgatgaaaa gggttctacg ggcagtgaag aattactacc agacaacaga 2520

gaaattttct tttggaagaa tttgacatat caagtcaaga ttaagaaaga agatagagtc 2580

attttagacc atgttgatgg ttgggttaaa ccaggtcaaa ttactgcatt gatgggtgca 2640

tctggtgctg gtaagaccac tttgttgaat tgtttatctg agagagtaac tactggtgtt 2700

attactgatg gtgtgagaat ggttaatggt catgcgttag attcttcgtt ccaaagatca 2760

attggttatg tgcaacaaca agatgttcat ttacagacat ctacagttag agaagcgttg 2820

caattctccg catatttgag acaatcaaac aaaatatcta agaaggagaa ggatgaatat 2880

gttgactacg tcattgactt gttggagatg actaactatg cggatgcatt ggttggtgtt 2940

gccggtgaag gtttgaatgt tgaacaaaga aagagattaa ccatcggtgt tgaattagtt 3000

gccaagccta agttgttact attcttggat gaaccaactt ctggtttaga ctcccaaact 3060

gcctggtcta tttgtaagtt gatgagaaag ttagctgatc atggtcaagc tatcttgtgt 3120

acaattcatc aaccttccgc acttattatg gctgaattcg atagattgtt gtttttgcaa 3180

aagggtggta gaactgctta ttttggtgac ttgggtaaaa actgtcaaac catgattgac 3240

tactttgaaa aacacggagc agatccatgt cccaaagaag ccaatccagc agaatggatg 3300

ttggaagttg ttggtgccgc tccaggctcc catgctaaac aggactattt tgaagtttgg 3360

agaaactctg acgaatatag agctgttcaa aatgaaatca cccatatgga aactgaatta 3420

gttaaattac caagagatga agatcccgaa gcacttttga aatacgctgc acccatttgg 3480

aaacaatatt tgcttgttag ttggagggcg attgtacaag attggagatc acctggatat 3540

atatactcca aatttttctt gattatcgtg tcatctatat tgattggatt ttcatttttt 3600

aaagccaaaa atacagttca agggttgacg aatcaaatgc ttgctatatt tatgttcaca 3660

gttcaattca caactattat tgaccaaatg ttgccatttt ttgttcgaca acgtgaggtg 3720

tatgaggtta gagaagcacc ttccagaaca tatagttggg ttgccttcat tacaggtcaa 3780

ataacttcag agcttcctta tcaaataatt gttggaacga ttgctttctt ctgctggtac 3840

tatcctgttg gattatatac caatgctgaa cctacacata gtgtgactga acgtggtgcc 3900

ttgatgtggt tgtttattac ttcatttttt gtttacacat caacatttgg tcaattatgt 3960

atgtcattca atgaagatat tgaaaatgct ggaactgttg ctgctacatt attcaccttg 4020

tgtttgatat tttgtggtgt tatggttgtt ccagagaata tgccacgatt ttggattttc 4080

atgtacagat gtaatccatt tacttatatg attcaaggtg ttctttcaac gggattagct 4140

cgcaataaag ttgtttgtgc tgcaagagaa cttgttctgc ttcaaccacc aaaaggtcaa 4200

acttgttctt cattcttgga tccttatatc agtgtggctg gaggttatta tttacctaat 4260

aatgatggaa cttgttcatt ctgttcagta gataatactg atatgttttt acatcgtatc 4320

catgccttat acagtgagag atggagaaat tttggattat ttattacatt cattgtgatt 4380

aatgttgtct tgactgtatt cttttattgg ttagctaggg taccaaaagg gtcaagatca 4440

aagactaaaa agtga 4455

<210> 4

<211> 1372

<212> DNA

<213> Candida tropicalis

<400> 4

aagagatttc ttgtattcat tacaattatt acatggttga aaactgtatc aactgctcat 60

attcctgcac cacttgctga tccaagtaga gatgagtttt atactccatc tccaggtttt 120

gaatacgcta ctccaggaac tattttaaaa atccgtccaa ctcctcgtgc tgttcgtaat 180

ttattattct ttcatgttcc tttaaaaaac tcttggcaat tgttggttag atctcaagat 240

tcttttggtg aacctaatgc tatagttact acaattcttg aacctatgaa ttcaaatcct 300

tcaaaaattt tatcttatca aacttttgaa gattcaactt cattaaaatg cgctaccagt 360

tataattatc aagttggtat tccaccattt ggaaatgttg ctacccaatt tgaaatgaaa 420

tttataattc ctgctttaaa taaaggatat tttgtaatta gtcctgatta tgaaggacca 480

agaggtgcat ttactgttgg tgcacaagca gcacatgcag tattggattc tattcgtgct 540

gtattgaatt ctgggtctat aacttctatt gatccagatg ctaaagttgc aatgtggggt 600

tattctggag gatccttagc atcaagttgg gcagctgtaa tgcaacctga atatgcacct 660

gaattgtcaa ataatttaat aggtgttgcc ttggggagga tttgttacta atataactgc 720

tgttgctgaa tattctgata gaactccact ttctggtctt gttccagtag cacttaatgg 780

attagccaat gaatatccat tggttagaca attgcttaat caagaaataa gtcctaaaaa 840

aaatgcaagt tttcatcgtg gagttcaaaa atgttttctt cctgctatag cttattttag 900

aggaagaact attcttggta gaaataatga aaagaaagca atgtttccta atggatggca 960

tttacttgat aatcccggat ttttttgaaa ttcttgataa aaataatttg atttcttata 1020

acgcaattcc aaaaattcca atatttgtat atcatggaac aaaaaagatg gagttgttcc 1080

gatttcctat gctcataaaa ttttcgataa atggtgtgat gagggaattg aatcgtttga 1140

atttgcagaa tctttaacta ctggacatat attggaaact tttactggtg ctgcagccgc 1200

ttggacttgg ttacaaaaac gatttgatga tgtacctcca tataatggtt gtttccatac 1260

aagaagactc actaatttga agtacacggg agcatcaaag agtataattg attattacga 1320

tgggttgttt aaagaaagct tcactgtgaa gaatagtacc tatcttgtct ag 1372

<210> 5

<211> 1960

<212> DNA

<213> Candida tropicalis

<400> 5

atgtcaggat tagaaattgc tgcagctgcc gttcttggta gtcagttatt agaagccaaa 60

tatttaattt ccgatgatgt actgttggcc aaaacagttg ctcttaatgc acttccatat 120

ttatggaaag cctccagggg taaagcttca tattggtatt tctttgaaaa atcagtattt 180

aaaaatccaa ataataaagc attggcattt ccaagaccaa gaaagaatgc accaccacca 240

aaggttgatg atgaaggatt tcaaatttat gacgatcaat ttgacctaga agaatatacc 300

tataaggaat tgtatgacat ggttttgaaa tactcttaca ttttgaaaca tgaatatggt 360

gttactgcaa atgatactat tggtgtttct tgtatgaata aaccactttt cattgtttta 420

tggttggcct tatggaatat tggtgccttg ccagcatttt tgaatttcaa caccaaagat 480

aaaccattga ttcactgtct taaaattgtc aatgctagtc aagttttcgt tgatcctgat 540

tgtgatgctc caatcaaaga tactgaatct caaattaaag aggaattacc acatgttaga 600

ataaattaca ttgatgaatt tgctttgtat tgatagatta agactcaagt ctactccaaa 660

atacagagct gaagatagta ctagaagacc aacagatacc gattcttccg cctgtgcgtt 720

gatctataca tcaggtacca ctggtttacc aaaagcaggt atcatgtctt ggagaaaagc 780

attcatggct tctgtttcct ttggccatat tatgaaaatt aagaatgatt ccaatgtttt 840

tacagttatg ccattgtatc attcaacagc tgctatgttg ggtttgtgtc ctactttaat 900

tgttggtggt tgtgtttctg tttctcaaaa attctcagcc acttcattct ggactcaagc 960

tagattatgt ggtgccacac atattcaata tgttggtgaa gtttgtcgtt atttgttaaa 1020

ctcaaaacat cacccagatc aagatagaca caatgttaaa attgcctatg gtaatggatt 1080

acgtccagat atatggtctg aattcaagag aagattccac attgaaggta ttggggaatt 1140

ttatgcagct actgaatctc caattgccac tacaaactta caatacggtg aatatggtgt 1200

aggtgcctgt cgtaaatatg gttcacttac tagtttattg ttatctaccc aacaaaaatt 1260

ggccaagatg gatccagaag atgaaagtga aatttataag gatccaaaaa ctggattttg 1320

tgttgaagct gcatataatg aacctggtga attgttgatg agaattttaa atcctaatga 1380

tattcaaaaa tcattccaag gttattatgg taacaaatct gctaccaata gcaaaattct 1440

cacgaatgtt ttcaaaaaag gagatgcttg gtatagaagt ggtgacttgt tgaaaatgga 1500

tgaacatcaa ttgttgtatt ttgttgatag attgggtgag aaataccttc cgttggaaat 1560

cagaaaatgt ttcagcaact gaagttgaaa atgagttgat gggatctaaa gcattgaaac 1620

aatctgttgt tgttggtgtt aaagttccag gaatcacgaa ggtagagctt gttttgctgt 1680

atgtgaagca aaagatgatt taactcatga agatattttg aaattgattc atggacatgt 1740

tactaaatcg ttaccagttt atgcacaacc tgcattcatt aaaatcggat ccattgaagc 1800

ttctcataat cataaagttc caaagaatca atttaagaat caaaaattac caaaaggtga 1860

agatggtaaa gacttgattt actggttgaa tggtgataaa tatcaagagt tgactgaaga 1920

ggattggtct ttgatctgta ctggtaaagc caaattgtaa 1960

<210> 6

<211> 4455

<212> DNA

<213> Candida tropicalis

<400> 6

atgggagaaa taaccccaac tgacaaaagc gaagaccagt caatggttaa tgcatatcat 60

ggatttgata ctcatgcatc agaagatata caagatttag ccaaaacttt tactcatcat 120

tcaattggcg atggtactga tggtttacaa agatatctta caaatatgac agaagtacca 180

ggtataaatc cttacaccga agatatttac actagtgacc aattgaatcc agactcagat 240

aattttaatg caaagttttg gatcaagaac ttgagaaaat tgtatgattc agatccagat 300

tattacaagc catcaagatt gggagttgcc tatagagatt taagagctta tggtgtggcc 360

aatgattctg attaccagcc cactgtggca aacgcggtct ggaagtttat caaagaggga 420

ttgcattatt tagaaaaagg tgatggctca aggtattttg atattttaaa atcaatggat 480

ggaataatga aaccaggtga acttacagtt gttttaggta gaccaggggc tggttgttcc 540

acattgttga aaacattggc ttcacaaaca tatggatttc atattggaaa agaatcaaaa 600

atcagttatg atggtttaac tcctcccgaa atcgaaaaaa cttatagggg taatgttgta 660

tactctgcag aaacagatgt tcattttcca catttgactg tcggacaagt cttggaattt 720

gctgctagaa tgagaacgcc acagaacaga ggtgaaggtg tagatagaga aacatatgcc 780

aaacaccatg ttagtgttta tatggctact tatgggttat ctcatacaag aaataccaat 840

gtgggtaacg attttgtcag aggagtttct ggtggtgaaa gaaaaagggt ctccattgct 900

gaagtttcgt tgagtggtgc aaacgttcaa tgttgggata atgccactaa aggtttggat 960

gctgcaaccg cattggaatt catcagagca ttgaagactt ctgctgctat tttggaaagt 1020

accccattga ttgctactta tcaatgttca caagatgctt atgacttgta tgataatgct 1080

gtcgttttgt atgaaggttt ccaaattttt tttggtaaag ccaataaagc caaggagtat 1140

tttgtaaaca tgggatacaa gtgtcctcat agacaaaaca ctgctgactt tttaacttca 1200

ttgactaatc cagctgaaag agagccatta ccaggttatg agaataaagt cccaaggact 1260

cctcaagaat ttgaagcata ttggaagaaa tccccagagt atactgcatt ggttaatgaa 1320

attcattcat atttcattga gtgtgagaaa ttaaacacca gacaactcta ccaagattca 1380

catgttgcaa gacaatccaa caatattcgt ccatcttcac catatactgt atcatttttc 1440

atgcaagtaa agtatgttat acaaagaaat ttcctccgta tgaaagctga tccatcgatt 1500

ccgttgacta ctattttctc acaactagtt atgggactta ttcttgcctc ggtattttac 1560

aatcttcctg caacttcagg ttctttttac taccgatccg gtgcgcttta ctttggtttg 1620

ttatttaatg ctatttcgtc cctacttgaa attattgccc tttttgaagc aagacccatt 1680

gttgagaaac ataaaaaata tgccctttat cgtccatcag cagatgcatt agcaagtatt 1740

ataagtgagt taccagttaa gttttttcaa tccttgtgtt tcaacattcc tttctatttt 1800

atggttaacc ttagaagaga tgctggtaga ttcttctttt attggttaat tggtatatta 1860

ggtacattca ttatgtcaca cttattcaga tctattggtg cagtatttac tactttagca 1920

ggtgctatga ctccggcggg ggtgatttta ttagcaatga tattatttgc tggatttgtc 1980

attccatttc caagcatgtt gggttggtct aaatggataa aatggataaa tcctgtcact 2040

tatttgtttg aatcacttat ggtaaacgag tatcataata gagagtttga atgcagtgat 2100

ttcgtaccta tgggaccagg atatgagaat cttagtcttg aaaataaggt ttgttcaagt 2160

ttgggtggca tccctggtag tgcttttgtt caaggtgatg attatttaag acttggattt 2220

gccttttcta actcccataa gtggagaaat tttggtatat ctgttgcgtt tgctgtgttt 2280

cttttgtttc tttatgttgc attgactgaa ctcaataaag gtgctatgca aaaaggtgaa 2340

attgtgttgt ttcttagagg atctttgaag aaatacaaga gaaactccag tagcgcagat 2400

attgaatccg gtaaagaaat agtgaaattt aatttccaag acgaagcaga atcttctaat 2460

agtgatcgta ttgatgaaaa gggttctacg ggcagtgaag aattactacc agacaacaga 2520

gaaattttct tttggaagaa tttgacatat caagtcaaga ttaagaaaga agatagagtc 2580

attttagacc atgttgatgg ttgggttaaa ccaggtcaaa ttactgcatt gatgggtgca 2640

tctggtgctg gtaagaccac tttgttgaat tgtttatctg agagagtaac tactggtgtt 2700

attactgatg gtgtgagaat ggttaatggt catgcgttag attcttcgtt ccaaagatca 2760

attggttatg tgcaacaaca agatgttcat ttacagacat ctacagttag agaagcgttg 2820

caattctccg catatttgag acaatcaaac aaaatatcta agaaggagaa ggatgaatat 2880

gttgactacg tcattgactt gttggagatg actaactatg cggatgcatt ggttggtgtt 2940

gccggtgaag gtttgaatgt tgaacaaaga aagagattaa ccatcggtgt tgaattagtt 3000

gccaagccta agttgttact attcttggat gaaccaactt ctggtttaga ctcccaaact 3060

gcctggtcta tttgtaagtt gatgagaaag ttagctgatc atggtcaagc tatcttgtgt 3120

acaattcatc aaccttccgc acttattatg gctgaattcg atagattgtt gtttttgcaa 3180

aagggtggta gaactgctta ttttggtgac ttgggtaaaa actgtcaaac catgattgac 3240

tactttgaaa aacacggagc agatccatgt cccaaagaag ccaatccagc agaatggatg 3300

ttggaagttg ttggtgccgc tccaggctcc catgctaaac aggactattt tgaagtttgg 3360

agaaactctg acgaatatag agctgttcaa aatgaaatca cccatatgga aactgaatta 3420

gttaaattac caagagatga agatcccgaa gcacttttga aatacgctgc acccatttgg 3480

aaacaatatt tgcttgttag ttggagggcg attgtacaag attggagatc acctggatat 3540

atatactcca aatttttctt gattatcgtg tcatctatat tgattggatt ttcatttttt 3600

aaagccaaaa atacagttca agggttgacg aatcaaatgc ttgctatatt tatgttcaca 3660

gttcaattca caactattat tgaccaaatg ttgccatttt ttgttcgaca acgtgaggtg 3720

tatgaggtta gagaagcacc ttccagaaca tatagttggg ttgccttcat tacaggtcaa 3780

ataacttcag agcttcctta tcaaataatt gttggaacga ttgctttctt ctgctggtac 3840

tatcctgttg gattatatac caatgctgaa cctacacata gtgtgactga acgtggtgcc 3900

ttgatgtggt tgtttattac ttcatttttt gtttacacat caacatttgg tcaattatgt 3960

atgtcattca atgaagatat tgaaaatgct ggaactgttg ctgctacatt attcaccttg 4020

tgtttgatat tttgtggtgt tatggttgtt ccagagaata tgccacgatt ttggattttc 4080

atgtacagat gtaatccatt tacttatatg attcaaggtg ttctttcaac gggattagct 4140

cgcaataaag ttgtttgtgc tgcaagagaa cttgttctgc ttcaaccacc aaaaggtcaa 4200

acttgttctt cattcttgga tccttatatc agtgtggctg gaggttatta tttacctaat 4260

aatgatggaa cttgttcatt ctgttcagta gataatactg atatgttttt acatcgtatc 4320

catgccttat acagtgagag atggagaaat tttggattat ttattacatt cattgtgatt 4380

aatgttgtct tgactgtatt cttttattgg ttagctaggg taccaaaagg gtcaagatca 4440

aagactaaaa agtga 4455

<210> 7

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> ADHpro_F

<400> 7

aaactcgagt ctagctccct aacatgtagg t 31

<210> 8

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> ADHpro_R

<400> 8

aaagtcgaca gttgattgta tgcttggtat agctt 35

<210> 9

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> ADHter_F

<400> 9

aaagtcgact ctagataagc gaatttctta tgatttatga ttttta 46

<210> 10

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> ADHter_R

<400> 10

aaagcggccg cgtgtggaag aacgattaca acag 34

<210> 11

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<223> LIP1_F

<400> 11

aaagtcgaca tgagatttct tgtattcatt acaattatta catggttgaa aac 53

<210> 12

<211> 61

<212> DNA

<213> Artificial Sequence

<220>

<223> LIP1_R

<400> 12

aaatctagag tggtggtggt ggtggtggac aagataggta ctattcttca cagtgaagct 60

t 61

<210> 13

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> FAT1_F

<400> 13

aaagtcgaca tgtcaggatt agaaattgct gcagctgcc 39

<210> 14

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> FAT1_R

<400> 14

aaatctagac aatttggctt taccagtaca gatcaaagac ca 42

<210> 15

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> MRP1_F

<400> 15

aaagtcgaca tgggagaaat aaccccaact gacaaaagcg 40

<210> 16

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> MRP1_R

<400> 16

aaatctagac tttttagtct tgaccctttt ggtacc 36

<210> 17

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> P1_F

<400> 17

aaaggatcct ctagctccct aacatgtagg t 31

<210> 18

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> P1_R

<400> 18

aaagtcgaca gttgattgta tgcttggtat agctt 35

<210> 19

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> P2_F

<400> 19

aaagtcgacc acgtgtctag ctccctaaca tgtaggt 37

<210> 20

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> P2_R

<400> 20

aaagcggccg cagttgattg tatgcttggt atagctt 37

<210> 21

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> P3_F

<400> 21

aaagtcgact aagcgaattt cttatgattt atgattttta 40

<210> 22

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> P3_R

<400> 22

aaacacgtgg tgtggaagaa cgattacaac ag 32

<210> 23

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> P4_F

<400> 23

cagttgattg tatgcttggt atagtcgaca tgagatttct tgtattcatt acaattatt 59

<210> 24

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> P4_R

<400> 24

gttaactaag cgaatttctt atgatttatg tcgacagtgg tggtggtggt ggtg 54

<210> 25

<211> 56

<212> DNA

<213> Artificial Sequence

<220>

<223> P5_F

<400> 25

cagttgattg tatgcttggt atagcgggcc gcatgtcagg attagaaatt gctgca 56

<210> 26

<211> 61

<212> DNA

<213> Artificial Sequence

<220>

<223> P5_R

<400> 26

gttaactaag cgaatttctt atgatttatg cggccgcaca atttggcttt accagtacag 60

a 61

<210> 27

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> P6_F

<400> 27

aaagcggccg cataagcgaa tttcttatga tttatgattt tta 43

<210> 28

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> P6_R

<400> 28

aaactcgagc acgtgagttg attgtatgct tggtatagct t 41

<210> 29

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> P7_F

<400> 29

aaacacgtgt aagcgaattt cttatgattt atgattttta 40

<210> 30

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> P7_R

<400> 30

aaactcgagg tgtggaagaa cgattacaac ag 32

<210> 31

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<223> P8_F

<400> 31

cagttgattg tatgcttggt atagcacgtg atgggagaaa taaccccaac tg 52

<210> 32

<211> 59

<212> DNA

<213> Artificial Sequence

<220>

<223> P8_R

<400> 32

gttaactaag cgaatttctt atgatttaca cgtgcttttt agtcttgacc cttttggta 59

<210> 33

<211> 56

<212> DNA

<213> Artificial Sequence

<220>

<223> P9_F

<400> 33

gcttgatatc gaattcctgc agcccggggg atcctctagc tccctaacat gtaggt 56

<210> 34

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> P9_R

<400> 34

ggggggcccg gtacccaatt cgccctctcg aggtgtggaa gaacgattac aacag 55

Claims (10)

1. Candida tropicalis (Candida tropicalis) strains with improved tolerance to substrate cytotoxicity,

wherein the strain comprises a mutation in one or more genes selected from the group consisting of: consisting of SEQ ID NO: 1, the LIP1 (lipase) gene represented by the base sequence shown in SEQ ID NO: 2 and a FAT1 (fatty acid transport) gene represented by the base sequence shown in SEQ ID NO: 3, MRP1 (multidrug resistance protein) gene represented by the base sequence shown in the above formula, or

Wherein the strain is transformed with one or more mutant genes selected from the group consisting of: a mutated LIP1 gene, a mutated FAT1 gene, and a mutated MRP1 gene.

2. The candida tropicalis strain according to claim 1, wherein the mutated LIP1 (lipase) gene is as set forth in SEQ ID NO: 4, respectively.

3. The Candida tropicalis strain according to claim 1, wherein the mutated FAT1 (fatty acid transport) gene is as set forth in SEQ ID NO: 5, respectively.

4. The candida tropicalis strain according to claim 1, wherein the mutated MRP1 (multidrug resistance protein) gene is as set forth in SEQ ID NO: and 6.

5. The Candida tropicalis strain according to claim 1, wherein the Candida tropicalis strain has a blocked β -oxidation pathway.

6. The Candida tropicalis strain according to any of claims 1 to 5, wherein the substrate is Fatty Acid Methyl Ester (FAME).

7. The Candida tropicalis strain of claim 6, wherein the fatty acid methyl ester substrate comprises a compound selected from C₆-C₂₀One or more fatty acid methyl esters.

8. A process for producing a dicarboxylic acid (DCA), the process comprising:

a Candida tropicalis strain as defined in any of claims 1 to 5 is cultured in a culture medium together with a substrate.

9. The method of claim 8, wherein the substrate is Fatty Acid Methyl Ester (FAME).

10. The method of claim 9, wherein the fatty acid methyl ester comprises a fatty acid selected from C₆-C₂₀One or more fatty acid methyl esters.

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